Nanoscale imaging of molecular positions and anisotropies

Abstract

A Polarization Fluorescence Photoactivation Localization Microscopy (P-FPALM) system and method are provided to simultaneously image the localizations and fluorescence anisotropics of large numbers of single molecules within a sample. The system modifies known FPALM systems by adding a polarizing beam splitter. The beam splitter polarizes emissions perpendicular and parallel to an axis in the sample to allow spatially separate imaging of fluorescence emitted from a sample. The system includes lenses and mirrors so that the separate, polarized beams are detected simultaneously. The present invention includes methods of using the system to image localizations and fluorescence anisotropics of single molecules, and methods of using data obtained with the system to predict 3-D orientation of the molecules. The system and method achieve substantially improved lateral resolution within even dense samples over known microscopic imaging techniques, and does not compromise speed or sensitivity.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a system and method of imaging at a nanoscale level. More particularly, the present invention is a system and method for imaging single molecule polarization anisotropy in biological specimens.BACKGROUND OF THE INVENTION[0002]For a complete understanding of cellular biology we ideally need to observe the cell at a molecular level. Single-molecule detection gives extra statistical information and allows the exploration of heterogeneity in samples, as well as direct observation of dynamic state changes arising from photophysics and photochemistry. For every distinct molecule it can be useful to know the number of its kind present, the precise location of each member of the ensemble of identical molecules, and the functionality of each member of the ensemble. Such observations can only be made by single molecule microscopy, since standard imaging methods, which make ensemble measurements that average over many molecules, do n...

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Benefits of technology

[0015]The ability to image anisotropy with resolution below the diffraction limit presents several interesting opportunities, most importantly the ability to image short-range order and to quantify the degree of preferential orientation of molecules. As long as the limitations of the method are taken into account, we can use the anisotropy to estimate the degree of alignment (but not the precise angle) between the transition-dipole moment of the emitting molecule (the fluorophore) and the excitation laser beam polarization. Interactions between membrane domains and the cytoskeleton, such as those found in focal adhesions, are expected to result in preferential orientation of molecules, but the size of those structures is generally well below the diffraction limit. Th

Problems solved by technology

Such observations can only be made by single molecule microscopy, since standard imaging methods, which make ensemble measurements that average over many molecules, do not provide quantitative information about each molecule.

Light microscopy provides non-invasive imaging of mult

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